Host apolipoprotein b messenger RNA-editing enzyme catalytic polypeptide-like 3G is an innate defensive factor and drug target against hepatitis C virus

Authors

  • Zong-Gen Peng,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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    • These authors contributed equally to this work.

  • Zhi-Yun Zhao,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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    • These authors contributed equally to this work.

  • Yan-Ping Li,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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    • These authors contributed equally to this work.

  • Yu-Ping Wang,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Lan-Hu Hao,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Bo Fan,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Yu-Huan Li,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Yue-Ming Wang,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Yong-Qiang Shan,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Yan-Xing Han,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Yan-Ping Zhu,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Jian-Rui Li,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Xue-Fu You,

    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Zhuo-Rong Li,

    Corresponding author
    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
    • Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
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    • fax: 86-10-63017302

  • Jian-Dong Jiang

    Corresponding author
    1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
    • Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
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    • fax: 86-10-63017302


  • Potential conflict of interest: Nothing to report.

Abstract

Host cellular factor apolipoprotein B messenger RNA (mRNA)-editing enzyme catalytic polypeptide-like 3G (hA3G) is a cytidine deaminase that inhibits a group of viruses including human immunodeficiency virus-1 (HIV-1). In the continuation of our research on hA3G, we found that hA3G stabilizing compounds significantly inhibited hepatitis C virus (HCV) replication. Therefore, this study investigated the role of hA3G in HCV replication. Introduction of external hA3G into HCV-infected Huh7.5 human hepatocytes inhibited HCV replication; knockdown of endogenous hA3G enhanced HCV replication. Exogenous HIV-1 virion infectivity factor (Vif) decreased intracellular hA3G and therefore enhanced HCV proliferation, suggesting that the presence of Vif might be an explanation for the HIV-1/HCV coinfection often observed in HIV-1(+) individuals. Treatment of the HCV-infected Huh7.5 cells with RN-5 or IMB-26, two known hA3G stabilizing compounds, increased intracellular hA3G and accordingly inhibited HCV replication. The compounds inhibit HCV through increasing the level of hA3G incorporated into HCV particles, but not through inhibiting HCV enzymes. However, G/A hypermutation in the HCV genome were not detected, suggesting a new antiviral mechanism of hA3G in HCV, different from that in HIV-1. Stabilization of hA3G by RN-5 was safe in vivo. Conclusion: hA3G appears to be a cellular restrict factor against HCV and could be a potential target for drug discovery. (HEPATOLOGY 2011;)

Human APOBEC3G (apolipoprotein B messenger RNA [mRNA]-editing enzyme catalytic polypeptide-like 3G, hA3G) belongs to the APOBEC superfamily, which covers at least 10 members sharing a cytidine deaminase motif (a conserved His-X-Glu and Cys-X-X-Cys Zn2+ coordination motif).1 Accumulated evidence shows that hA3G in human lymphocytes represents an innate immunity factor against human immunodeficiency virus type 1 (HIV-1). It suppresses the HIV-1 replication cycle by incorporating into HIV-1 particles a cytidine deamination reaction in minus-strand complementary DNA (cDNA) leading to G to A hypermutated proviruses,2 and/or by inhibiting the process of reverse transcription.3, 4 To defeat the effect of host hA3G, HIV-1 develops an offensive device, called accessory protein Vif (virion infectivity factor). Vif binds to hA3G in the cytoplasm, forms the Vif-Cul5-SCF complex which drives host hA3G into a degradation process through the ubiquitine proteosome pathway (UPP) system, and thus effectively abolishes the anti-HIV activity of hA3G.5, 6 Interruption of the Vif-hA3G interaction has recently become a novel strategy for drug discovery against HIV-1.7, 8

In continuation of our research on hA3G, we synthesized a group of hA3G stabilizing compounds and found by chance that hA3G stabilizers have a significant anti-HCV effect. This provoked our strong curiosity for the role of host hA3G in HCV infection and for its translational potential. In fact, hA3G is reported to be a host restriction factor for a group of viruses including human HIV-1, T-cell leukemia virus type 1 (HTLV-1), hepatitis B virus (HBV), and parvoviruses.2, 3, 4, 9, 10, 11 It created great attention in the field of antiviral research. As current anti-HCV chemotherapy is far from satisfactory in the clinic, new mechanism drugs for hepatitis C is highly desirable.12 The goal of this study was to learn whether hA3G is a host innate immunity factor against HCV, and if so what is its potential as a drug target against HCV.

Abbreviations

APOBEC3G, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G; BUN, blood urea nitrogen; CC50, 50% cytotoxic concentration; CRE, creatine; EC50, half maximal effective concentration; GOT, glutamate-oxaloacetate transaminase; GPT, glutamate-pyruvate transaminase; hA3G, human APOBEC3G; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV-1, human immunodeficiency virus type 1; HTLV-1, T-cell leukemia virus type 1; IC50, half maximal inhibitory concentration; PEG-IFN, pegylated-interferon; UPP, ubiquitine proteosome pathway; Vif, virion infectivity factor.

Materials and Methods

Cells.

Huh7.5 human liver cells (kindly provided by Vertex Pharmaceuticals, Boston, MA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, CA) supplemented with 10% inactivated fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Invitrogen). GS4.3 cells (one of the human hepatoma Huh7 cells carrying an HCV subgenomic replicon I 377-3′del.S)13 was maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin-streptomycin (Invitrogen), and 500 μg/mL of geneticin (G418; Invitrogen). CEM-SS cells were from the American Type Culture Collection. Cells were cultured at 37°C in 5% CO2.

Agents.

Agent RN-5 (N,N′-(dimethylbipheny1-4, 4′-diyl) dibenzenesulfonamide, C26H24N2O4S2, molecular weight [MW]: 492.61)7 and IMB-26 ([3-(α-bromopropyl) amino-4-methoxy benzene] formyl (3′,4′,5′-trimethoxybenzene) amide, C20H23N2O6Br, MW = 467.31)8 were synthesized in the Medicinal Chemistry Laboratory of the Institute of Medicinal Biotechnology Chinese Academy of Medical Sciences, with a purity over 99.0%. The compound structure was confirmed with 1H-NMR and MS spectra. Interferon-α-2b (Intron A) was from Schering Plough (Brinny) (Kenilworth, NJ). BILN2061, a known NS3-4A protease inhibitor, was provided by Shanghai Lechen International Trading (Shanghai, China).

Plasmids.

Plasmid pcDNA3.1-Vif coding for HIV-1 full-length Vif was created by insertion of Vif (amplified by polymerase chain reaction [PCR] from HIV-1 plasmid SVC21.BH10) into pcDNA3.1; the plasmids hA2, hA3B, hA3C, hA3F, and hA3G that express wildtype forms of hA2, hA3B, hA3C, hA3F, and hA3G, respectively, possess a fused HA tag at the C-terminus. The above-mentioned plasmids were gifts from Dr. Shan Cen at the Lady Davis Institute for Medical Research and McGill University AIDS Centre. The plasmid pFL-J6/JFH/JC1 containing the full-length chimeric HCV cDNA was kindly provided by Vertex Pharmaceuticals (Boston, MA).

HCV RNA Synthesis, Transfection, and Generation of HCV Viral Stock.

Production of infectious HCV in hepatocytes was done as described.14 The plasmid pFL-J6/JFH/JC1 was restricted with XbaI and treated with Mung Bean nuclease (New England Biolabs) to generate the according HCV cDNA with T7 promotor. The cDNAs were purified and used as templates for RNA synthesis. HCV RNA was synthesized in vitro using a MEGAscript T7 kit (Ambion). The synthesized RNA was treated with DNase I (New England Biolabs) and purified with Wizard SV Gel and PCR Clean-Up System (Promega). The synthesized HCV RNA was used to transfect naïve Huh7.5 cells with the addition of Lipofectamine 2000 (Invitrogen). The culture medium was collected and cleaned with centrifugation at 3,000 rpm for 10 minutes. The supernatants were stored at −70°C as HCV viral stock and quantified with the Diagnostic Kit for Quantification of Hepatitis C Virus RNA (Shanghai Kehua Bio-Engineering).

Effect of Exogenous Human APOBECs and Vif on HCV Replication in Huh7.5 or in GS4.3 cells.

Huh7.5 cells 24 hours after HCV infection with viral stock (45 IU per cell) were transfected with different concentrations of APOBEC- or Vif-containing plasmids in the FuGENE HD Transfection Reagent (Roche), with pcDNA3.1 as plasmid control. Then, 72 hours later the culture medium was removed and total intracellular proteins were extracted using CytoBuster Protein Extraction Reagent (Novagen) with 1 mM protease inhibitor cocktail (Roche Applied Science). HCV Core, NS3, and hA3G protein (or APOBEC proteins with HA tag) was detected with western blot. A similar procedure was used for the experiment using GS4.3 cells, except the infection step.

Specific RNA Interference for hA3G in Huh7.5 Cells Acute Infection with HCV.

Huh7.5 cells were planted into the 6-well plate with 3 × 105 cells per well in the complete growth medium and infected with HCV viral stock (45 IU per cell). After 48 hours incubation, three hA3G gene-specific small interfering RNA (siRNA) sequences (5′-auuuaggcagauuauuccaaggcuc-3′, 5′-ucacgaugcagcuuccuccacuugc-3′, or 5′-uuuauguggagccugguugcauaga-3′; Invitrogen) were transfected into the cells using Lipofectamine RNAiMAX (Invitrogen), with a nonrelevant control siRNA as a reference. Cells were harvested for RNA extraction 48 hours after the transfection. Total RNA was analyzed for hA3G mRNA and HCV RNA expression using real-time reverse-transcription polymerase chain reaction (RT-PCR). The primers 5′-GAGCGCATGCACAAT GAC-3′ and 5′-GCCTTCAAGGAAACCGTGT-3′ were for hA3G mRNA, primers 5′-ACCCACTCCTCC ACCTTTG-3′ and 5′-CTGTAGCCAAATTCGTTGT CAT-3′ were for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA, and 5′-CGGGAGAGCCATA GTGGTCTGCG-3′ and 5′-CTCGCAAGCACCCTAT CAGGCAGTA-3′ for HCV RNA.15

Stabilization of hA3G with RN-5 or IMB-26 in Huh7.5 Cells.

The Huh7.5 cells were treated or untreated with RN-5 or IMB-26 at different concentrations for 24 hours. Total intracellular proteins were extracted using CytoBuster Protein Extraction Reagent with 1 mM protease inhibitor cocktail. The hA3G and actin protein were detected with western blot.

HCV Infection and Treatment in Huh7.5 Cells.

The Huh7.5 cells were seeded into 6-well plates (Costar) at a density of 3 × 104 cells/cm2. After 6 hours incubation, cells were infected with HCV viral stock (45 IU per cell) and simultaneously treated with RN-5, or IMB-26, or solvent control. The culture medium was removed after 96 hours inoculation and the intracellular RNA was extracted with RNeasy Mini Kit (Qiagen). Total intracellular proteins were extracted as well using CytoBuster Protein Extraction Reagent (Novagen) with 1 mM protease inhibitor cocktail (Roche Applied Science). The intracellular HCV RNA was quantified with a one-step RT-PCR kit (Invitrogen). HCV Core and hA3G protein was detected with Western blot.

Purification of HCV Virions.

Ten days after HCV infection the Huh7.5 cells were planted into 100 mm tissue culture dish (BD #353003) at a density of 3 × 104/cm2, followed by addition of RN-5, or IMB-26, or the solvent. After treatment for 96 hours, culture supernatants were collected and centrifuged at 3,800 rpm for 10 minutes, then layered onto a 20% sucrose cushion in TNE (10 mM Tris, 150 mM NaCl, 2 nM ethylene diamine tetraacetic acid) and ultracentrifuged at 250,000g for 3 hours at 4°C. Viral pellets were then resuspended in TNE, and the HCV Core and hA3G protein in the HCV viral particles were detected with western blot.

Effect of RN-5 or IMB-26 on Viral RNA Hypermutation.

Ten days after HCV infection the Huh7.5 cells were planted into 6-well plates at a density of 12 × 104/cm2, followed by addition of RN-5, or IMB-26, or the solvent. After treatment for 48 hours, culture supernatants were collected and centrifuged at 3,800 rpm for 10 minutes and the HCV RNA in the supernatant was extracted with RNeasy Mini Kit (Qiagen). In the same stage, naïve Huh7.5 cells were infected with the above-mentioned supernatants. After 8 hours infection, intracellular HCV RNA was extracted with RNeasy Mini Kit (Qiagen). The supernatant and intracellular HCV RNA (sited in the region of 5′-UTR and core) were amplified with the sense primer 5′-GAAT CACTCCCCTGTGAGGAAC-3′ and antisense primer 5′-CATTGGTGCAGTCGTTAG-3′. The PCR products were purified with Wizard SV Gel and PCR Clean-Up System (Promega) and inserted into the pGEM-T easy system II (Promega), followed by transfecting the conjunct vector into a sensitive bacterial of JM109 (Promega) and cultured in the Luria Broth solid culture media (Invitrogen) with 100 μg/mL ampicillin. The clone was selected and amplified. The plasmids were extracted with QIAprep Spin Miniprep Kit (Qiagen) and sequenced at Invitrogen (China).

Treatment of HCV in GS4.3 Cells.

The GS4.3 cells were seeded into 96-well plates (Costar) at a density of 3 × 104/cm2. After 6 hours incubation, cells were treated with RN-5, or IMB-26, or solvent; 96 hours later the intracellular RNA was extracted and HCV RNA was quantified with real-time RT-PCR. The half maximal effective concentration (EC50) was calculated with Reed & Muench methods.

Cytotoxicity Assay.

The Huh7.5 and GS4.3 cells were used in the test; 100 μL of 1 × 105/mL cells were planted into the 96-microwell plates. Six hours later the culture media were replaced with fresh medium containing RN-5 or IMB-26 at various concentrations. Cytotoxicity was evaluated with the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 96 hours. The 50% cytotoxic concentration (CC50) was calculated with Reed & Muench methods.

HCV NS3-4A Protease Assay In Vitro.

The assay was conducted in Buffer A containing 30 mM NaCl, 5 mM CaCl2, 10 mM DTT, 50 mM Tris (pH 7.8) using the Ac-Asp-Glu-Asp(EDANS)-Glu-Glu-Abu-Ψ-[COO]-Ala-Ser-Lys (DABCYL)-NH2 (FRET-S) fluorescent peptide (AnaSpec, USA) as substrate. Briefly, 140 μL buffer A, 20 μL compounds dissolved in buffer A with different concentration, and 20 μL HCV NS3-4A protease diluted in buffer A were added into 96-well plates and mixed well. The reaction was initiated by adding 20 μL of FRET-S. Reactions were continuously monitored at 37°C using a BMG Polarstar Galaxy (MTX Lab Systems, USA) with excitation and emission filters of 355 nm and 520 nm, respectively.

Real-Time qRT-PCR.

Total RNA extracted from cells was analyzed with SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen). Fluorescent signal was detected with iQ5 PCR detection system (Bio-Rad).

The primer pairs of 5′-CGGGAGAGCCATAGT GGTCTGCG-3′ and 5′-CTCGCAAGCACCCTATC AGGCAGTA-3′ were for HCV RNA,15 and 5′-CGG AGTCAACGGATTTGGTCGTAT-3′ and 5′-AGCC TTCTCCATGGTGGTGAAGAC-3′ were for GAPDH RNA.

Western Blot.

The extracted total protein or viral lysates were denatured by adding 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (250 mM Tris-HCl, pH 6.8, 5% dithiothreitol, 10% SDS, 0.5% bromophenol blue, 50% glycerol), followed by boiling for 5 minutes at 100°C. Proteins were analyzed with SDS-PAGE, then transferred onto nitrocellulose membranes (GE Healthcare) using Electroblotter (Bio-Rad). The membranes were blocked with 5% nonfat dry milk in the TBS-T (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 hour and washed 3 times in TBS-T 10 minutes each. Membrane samples were probed with monoclonal antibody specific for HCV core or NS3 (Abcam), or hA3G (Abcam), or HA protein (Santa Cruz Biotechnology). As a control, polyclonal antibody to actin (Santa Cruz Biotechnology) was used. After washing with TBS-T, the membranes were respectively incubated with secondary antibody of goat antimouse (for HCV core or NS3), goat antirat (for hA3G or HA), or goat antirabbit (for actin) (ZSGB-BIO, China) at room temperature for 1 hour. Protein signal was detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) with Alpha Innotech Focus and Image Acquisition (Alpha Innotech). Density scanning was done for semiquantification.

Immunofluorescent Staining.

The Huh7.5 cells at a density of 3 × 104 cells/cm2 were seeded into 24-well plates with a 13-mm diameter coverslip. After 6 hours incubation, cells were infected with HCV viral stock (45 IU per cell) and simultaneously treated with RN-5 or IMB-26. The cells were incubated for another 96 hours and then washed twice with ice-cold phosphate-buffered saline (PBS), fixed in paraformaldehyde for 10 minutes, and permeabilized with PBS containing 0.25% Triton X-100 for 5 minutes. Cells were next blocked in TBS containing 5% bovine serum albumin (BSA)/0.1% Tween-20, followed by an overnight treatment with anti-hA3G and anti-HCV core antibodies at 4°C. After 3 washes in TBS with 0.1% Tween-20, cells were probed with goat antirat Cy3 (Beyotime) and goat antimouse Dylight488 (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 1 hour. Then the slides were washed 3 times. Cell nuclei were counterstained with Hoechst 33342 (Beyotime) for 5 minutes at room temperature. Slides were mounted with antifade mounting medium and visualized using a Leica TCS SP2 laser scanning spectral confocal microscope. CEM-SS cells that are null of endogenous expression of hA3G was used as negative control.

Safety Measurement In Vivo.

Male and female Kunming mice (4 weeks, weight 18 ± 1.0 g) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China). They were fed with regular rodent chow and housed in an air-conditioned room. The mice were randomly divided into five groups with 10 mice each (five male plus five female). RN-5 was given once intraperitoneally (0, 62.5, 125, 250, or 500 mg/kg) or orally (0, 125, 250, 500, or 1,000 mg/kg). Body weight as well as survival was monitored. Blood samples were taken for liver and kidney function examination after 7 days treatment. The 0.3% carboxymethylcellulose sodium was used as solvent for oral administration and 0.9% saline with 3% Tween-80 was for intraperitoneal injection.

Results

hA3G Is a Host Restriction Factor Against HCV.

We first examined whether addition of external hA3G would reduce HCV replication in the Huh7.5 cells. To introduce hA3G, HCV-infected Huh7.5 cells were transfected with hA3G expression vectors fusing HA tag at the C-terminus.16 As shown in Fig. 1A, with a dose-dependent increase of the expression of external hA3G-HA or total intracellular hA3G in the HCV-infected Huh7.5 cells, the intracellular HCV replication decreased. HCV core protein level was significantly lower in the cells transfected with external hA3G as compared to those without transfection of external hA3G. The experiment was repeated for over five times. It suggests that hA3G might be a defensive factor for HCV replication. Specific siRNAs were then used to silence the endogenous hA3G gene in Huh7.5 cells. Treatment of the HCV-infected Huh7.5 cells with specific hA3G siRNA (25 basepairs) reduced hA3G mRNA by 77% in the real-time RT-PCR assay; accordingly, intracellular HCV RNA load increased by ≈90% (Fig. 1B, upper). RNAi treatment with another siRNA sequence specific for hA3G showed a similar effect (data not shown). Accordingly, the intracellular HCV core protein level increased in parallel with the decrease of intracellular hA3G protein (Fig. 1B, lower). The results again indicated that intracellular hA3G is a host innate defensive factor against HCV. Similar to that in HIV-1 infection,17, 18 separate transfection of the HCV-infected Huh7.5 cells with other APOBEC3 family members (such as hA3C and hA3F) showed strong inhibitory effect on HCV replication as well (Fig. 1C).

Figure 1.

Effect of exogenous human APOBEC's members or HIV-1Vif on HCV replication in the HCV-infected Huh7.5 cells. (A) Inhibiting HCV core protein by transfecting HCV-infected Huh7.5 cells with exogenous hA3G tagged with HA. (B) Enhancement of HCV replication (real-time RT-PCR, upper; western blot, lower) by treating HCV-infected Huh7.5 cells with RNAi specific for hA3G; the calibration was done with housekeeping gene GAPDH (RNA) or actin (protein). (C) Inhibition of HCV by transfecting HCV-infected Huh7.5 cells with exogenous human APOBEC's members tagged with HA. (D) Transfection of the HCV-infected Huh7.5 cells with exogenous HIV-1 Vif decreased hA3G and therefore enhanced HCV replication.

HIV-1 accessory factor Vif is a 190-240 amino acid protein required for HIV-1 to replicate in hA3G-containing host cells.19, 20 It binds to hA3G in host cell cytoplasm and triggers hA3G ubiquitination and subsequent degradation by way of proteasomes. It is one of the most important mechanisms for HIV-1 to escape from cellular defensive factor hA3G. To verify the role of hA3G in HCV infection, external HIV-1 Vif was introduced into the HCV-infected Huh7.5 cells using the transfection plasmid described above. As shown in Fig. 1D, after adding plasmid pVif into HCV-infected Huh7.5 cells, the Vif protein expressed and hA3G significantly reduced in a dose-dependent manner; as a result, HCV replication increased. It appeared that the expression of transfected HIV-1 Vif caused a clearance of hA3G in the host cells, and generated an environment that favored HCV replication. The result provides another support for the observation that hA3G is a host defensive factor for HCV, and demonstrates that the presence of HIV-1 Vif protein in host cells might help HCV proliferation. It was estimated that 15%-30% of all HIV-infected persons are coinfected with HCV21; the molecular mechanism for the high incidence of HCV infection in HIV-positive individuals remains unclear. The results presented in Fig. 1 might help us to understand why HIV/HCV coinfection is common in HIV-1(+) individuals.

Stabilizing Host hA3G Inhibits HCV Replication.

If the above finding is true, then stabilization of hA3G should inhibit HCV replication. Thus, agents with a protective effect on hA3G were employed in the study. RN-5 (Fig. 2A, left) is a compound that protects hA3G from Vif-mediated degradation in 293T cells cotransfected with hA3G and HIV-1 Vif vectors.7 Here, RN-5 was used as a chemical probe to validate the role of hA3G in HCV replication. RN-5 treatment showed no toxicity in the Huh7.5 cells at the study doses (not shown), but significantly elevated the level of hA3G protein in naïve Huh7.5 cells after 24 hours treatment (Fig. 2B, upper), suggesting that RN-5 is active in stabilizing hA3G independent of Vif. The mechanism remains unknown. As IMB-26 protected hA3G by binding to hA3G,8 it could be a clue for future study. RN-5 exhibited an hA3G protection effect in the HCV-infected Huh7.5 cells as well (Fig. 2C, upper), parallel with which was a reduction of HCV core protein to a level almost undetectable when RN-5 concentration was up to 1.1 μg/mL (Fig. 2C, upper). Fluorescent immunostaining was done in the RN-5-treated HCV(+) Huh7.5 cells to visualize the in situ change. As shown in Fig. 2D, RN-5 treatment increased the hA3G level (red), and accordingly decreased intracellular HCV core protein signal (green). The activity of RN-5 on HCV replication was dose-dependent (Fig. 2E, lower). The EC50 of RN-5 on HCV was 0.34 μg/mL (or 0.69 μM; Fig. 2E, lower) and CC50 in the MTT assay was over 60 μg/mL (122 μM; Fig. 2E, upper), resulting in a therapeutic index of over 176. Anti-HIV compound IMB-26 (Fig. 2A, right) is another new hA3G stabilizer; it blocked the interaction between hA3G and Vif by way of a direct binding at hA3G and therefore protected hA3G from degradation.8 IMB-26 also increased intracellular hA3G in Huh7.5 cells (Fig. 2B, lower; Fig. 2C, lower) and accordingly inhibited HCV (Fig. 2C, lower; Fig. 2E). These results indicate that stabilization of host hA3G is an effective and a novel approach to suppress HCV replication. As a nucleic acid sequence homologous to that of Vif gene was not found in HCV in GenBank, the hA3G degradation mechanism in HCV infection remains to be clarified.

Figure 2.

Inhibitory effect of hA3G stabilizer RN-5 or IMB-26 on the HCV replication. (A) Chemical structure of RN-5 ((N,N′-(dimethylbipheny1-4,4′-diyl) dibenzene-sulfonamide) (left) and IMB-26 ([3-(α-bromopropyl) amino-4-methoxy benzene] formyl (3′,4′,5′-trimethoxybenzene) amide) (right). (B) Intracellular hA3G protein level elevated dose-dependently after 24 hours treatment with RN-5 (upper) or with IMB-26 (lower) in naïve Huh7.5 cells. (C) Intracellular hA3G protein increased and HCV core protein decreased after 96 hours treatment with RN-5 (upper) or IMB-26 (lower) in HCV-infected Huh7.5 cells. (D) Immunofluorescent staining detected the increase of intracellular hA3G (red signal) and the reduction of HCV core (green signal) after RN-5 (0.37 μg/mL) treatment of the HCV-infected Huh7.5 cells (middle and lower); blue signal, nucleus. CEM-SS cells that are null of endogenous hA3G expression served as negative control (upper). (E) Cytotoxicity of RN-5 or IMB-26 in Huh7.5 cells was tested with MTT methods (upper); and inhibitory activity of the compounds on intracellular HCV RNA was examined with real time RT-PCR and calibrated with housekeeping gene GAPDH (lower).

Anti-HCV Action Mode of hA3G Stabilizing Compounds.

To ensure the hA3G-mediated anti-HCV mechanism for the compounds (RN-5 and IMB-26), their activity on HCV viral enzymes was examined using the human GS4.3 cell line. The cell line was derived from the Huh7.0 cells transfected with HCV subgenomic replicons containing NS3, NS4A, NS4B, NS5A, and NS5B genes.13 These nonstructural genes cover almost all of the HCV replication enzymes; inhibiting any one of the enzymes terminates the replicon amplification.13 As shown in Fig. 3A, neither RN-5 nor IMB-26 inhibited amplification of the HCV replicons, indicating that the compounds were not inhibitors for any of the HCV enzymes. In this assay, interferon-alpha (Intron A) was used as a positive control, although its mechanism is not clear. The compounds were further tested in our cell-free HCV protease assay (NS3-4A), in which RN-5 and IMB-26 showed almost no inhibitory effect on the protease, even when the compound concentration was above 100 μg/mL (Fig. 3B). BILN2061, a known NS3-4A protease inhibitor, served as a positive control in the experiment. The experiment was repeated over three times.

Figure 3.

The mode of action of RN-5 or IMB-26 on HCV replication. (A) RN-5 and IMB-26 had no inhibitory activity on intracellular HCV replicon RNA in the GS4.3 cells; intron A was used as a positive control in the experiment. (B) RN-5 and IMB-26 had no inhibitory activity on HCV NS3-4A protease in vitro; BILN2061 was a positive control. (C) Transfection of GS4.3 cells with exogenous hA3G tagged with HA showed no effect on HCV amplification. (D) Virion encapsidation of host hA3G protein (intraviral hA3G) was increased in the supernatant HCV particles released from the RN-5 treated HCV-infected Huh7.5 cells. The first lane from left is the negative control for intraviral proteins.

Meanwhile, external hA3G with HA tag at the C-terminus was transfected into the GS4.3 cells. As shown in Fig. 3C, addition of external hA3G significantly increased the level of total intracellular hA3G in the GS4.3 cells in a dose-dependent manner; however, amplification of the HCV replicon (indicated by NS3) was not affected, suggesting that hA3G was without effect on the HCV enzymes. This agrees with the results from the hA3G stabilizers (Fig. 3A).

Next, we investigated whether RN-5 (or IMB-26) treatment increases the incorporation of hA3G into HCV viral particles. In this experiment, HCV-infected Huh7.5 cells were cultured for 2 days in the presence or absence of the hA3G stabilizers. The resultant HCV viral particles in the culture were harvested with ultracentrifugation. The hA3G protein within the viral particles was measured using western blot. As compared to those from untreated cells, the hA3G protein significantly increased in the HCV particles produced from the RN-5-treated HCV-infected Huh7.5 cells (Fig. 3D). Similarly, the increase of hA3G in HCV particles was also detectable after IMB-26 treatment (not shown). We assumed that the compounds might inhibit HCV through hA3G-mediated G/A viral mutation. To verify this, HCV genome sequencing was conducted for the supernatant viral particles released from HCV-infected Huh7.5 cells treated with RN-5 or IMB-26, as well as for the viruses that had already replicated in naïve Huh7.5 cells after infection with the HCV-containing supernatant mentioned above. The sequencing results, however, did not support our speculation. With respect to those from the cells without treatment, G/A mutation rate did not increase in the HCV viral particles generated from the RN-5 (or IMB-26)-treated Huh7.5 cells or in the newly replicated HCV after entering into Huh7.5 cells (Table 1). To confirm this result, a sensitive technique was used in which denaturation temperature was set at levels below 95°C.22 The results agreed with those in Table 1. Our studies show that hA3G might inhibit HCV replication through a mechanism different from that in HIV-1. As the antiviral mechanism of hA3G is complicated and varies among viruses,11, 23-28 more investigation is needed for the APOBEC superfamily in their action against HCV.

Table 1. Effect of RN-5 and IMB-26 on Viral RNA Hypermutation
SamplesTotal BasesTotal MutationsMutant Rate (‰)G to A Mutations
  • *

    Total number of HCV clones tested in the study.

  • The HCV RNA was from virus that had already entered into naïve Huh7.5 cells after infection with the supernatant viral particles released from HCV (+) Huh7.5 cells treated with RN-5 or IMB-26.

  • The HCV RNA was from the supernatant viral particles released from HCV (+) Huh7.5 cells treated with RN-5 or IMB-26.

IntracellularVirus control23920 (520 × 46*)361.515
RN-5 treated24440 (520 × 47*)281.156
IMB-26 treated24440 (520 × 47*)351.432
In supernatantVirus control8320 (520 × 16*)91.082
RN-5 treated2600 (520 × 5*)51.920
 IMB-26 treated9360 (520 × 18*)101.071

hA3G Stabilizers RN-5 and IMB-26 Are Safe In Vivo.

To evaluate the in vivo safety in stabilizing hA3G, RN-5 was given once to normal healthy mice orally (po) or intraperitoneally (ip), followed by body weight monitoring and organ function examination. After 7 days follow-up we found that RN-5, at doses between 125 and 1,000 mg/kg for oral administration or 62.5 and 500 mg/kg for ip administration, did not cause animal death or body weight change (Fig. 4A). Blood samples were taken for liver and kidney function examination at the end of the 7-day experiment. As shown in Fig. 4B, abnormality was not found in blood glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), blood urea nitrogen (BUN), or creatine (CRE) after RN-5 administration by the oral or ip route at the maximum dose used in the experiment. Considering the good safety of IMB-26 described in our previous report,8 we conclude that stabilization of hA3G might be a safe strategy for antiviral therapy.

Figure 4.

Safety of RN-5 in mice. (A) RN-5 ip injection or oral administration showed no effect on mice in survival rate and body weight (n = 10 for each dose group); (B) RN-5 also showed no effect on mice liver and kidney functions even at the maximum dose used in the study (500 mg/kg in ip and 1,000 mg/kg in oral) (n = 10). GOP, glutamate-oxaloacetate transaminase (U/L); GPT, glutamate-pyruvate transaminase (U/L); BUN, blood urea nitrogen (mM); CRE, creatine (μM).

Discussion

HCV is a single-stranded RNA virus belonging to the Flaviviridae family and is a causative agent for hepatitis C in the clinic. Combination of pegylated-interferon (PEG-IFN) with ribavirin is currently the conventional treatment for HCV infection.29, 30 However, the regimen is effective in only 40%-50% of patients infected with HCV genotype 1 and causes side effects.30, 31 Telaprevir and boceprevir have shown great promise in hepatitis C patients.32-34 However, these NS3/4A protease inhibitors caused drug-resistance.35-38 Clinical studies in hepatitis patients have shown that hA3G expression increased in HCV infection as well as in HBV/HCV coinfection,39, 40 but its role in HCV infection is unknown. Here for first time we have identified liver hA3G protein to be a host innate immunity factor for HCV infection. The action mode study using hA3G stabilizer RN-5 and IMB-26 revealed that the antiviral mechanism of hA3G for HCV appeared to be quite different from that for HIV-1. A related mechanism study is actively ongoing in our laboratories. For its potential in therapeutics, hA3G-mediated host antiviral machinery could be employed as a new strategy to discover broad-spectrum antiviral drugs for at least HCV, HIV-1, as well as HCV/HIV-1 coinfection. Furthermore, a combination of the NS3/4A protease inhibitor with hA3G stabilizers might generate an improved efficacy in the treatment of HCV infection and prevent development of drug resistance in HCV.

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